Transcript Presentations from our group - Sam Houston State University

Radhika Burra, Gonzalo A. Pradenas,
Claudio C. Vásquez and Thomas G. Chasteen
Selenium

identified as an element in 1917, named from the Greek
word, ‘selene’

exists in different forms:

metallic,

water soluble and

gaseous.

considered as an essential trace nutrient

used in the treatment of serious deficiency diseases

used as an anti-oxidant, in glass manufacturing industry, semiconductor materials and in electronic applications
Tellurium

discovered in 1782, named after Latin word ‘tellus’

extremely rare element

chemically related to selenium and sulfur

mildly toxic, teratogenic

used in semiconductor and electronic industry

used in the treatment of syphilis and leprosy
Why we are concerned???

exposure is fatal to living beings.
 considered as a severe environmental problem.
 environmental problems include,
 water contamination
•
•
Kesterson Reservoir of California
Power River Basin, Wyoming
 soil contamination
•
selenium contamination affecting plants and animals
What is Bioprocessing????
 Environmental clean-up method includes:



Biological treatment-bioprocessing.
Filtration after pH adjustment
Evaporation and soil removal Chemical detoxification methods
}
 Bioprocessing: also called bioremediation/bioreduction:
•
•
use of microorganisms or their enzymes for detoxification.
different microbial pathways for the metabolism of toxic
compounds.
• detoxify soluble toxic ions to insoluble and other less toxic
forms.
Bacteria currently being used
 LHVE - species of interest.
 characteristics include:



gram positive, rod shaped bacteria
forms spores.
gelatinase activity.
 classified as a Bacillus spp.
 isolated from Huerquehue National park, Chile.
 selenium (Se) resistant.
 reduce Se in solution to elemental Se.
 can be seen as a blood-red precipitate.
Chemical species of interest
 Anions of selenium:
 selenite (SeO32-)
 selenate (SeO42-)
 selenocyanate (SeCN-)
 Oxyanions of tellurium:
 tellurite (TeO32-)
 tellurate (TeO42-)
Instrumentation
 Gas chromatography with fluorine induced sulfur
chemiluminescence detector (GC-SCD)
 analyze and separate volatile compounds
 specific for Se, Te, and Sb compounds
 detection limits are in picogram range
 Gas chromatography- mass spectrometry (GC-MS)
 identification of structure of the unknown compounds
Sample preparation
 Luria-Bertani (LB) medium: tryptone, sodium chloride, yeast
extract, water.
 pH adjusted to 7.
 autoclave at 1200C.
 preparation of preculture.
 incubation at 370C for approximately 24 hrs.
 growth curve and headspace samples preparation.
 amendment with different metalloid concentrations.
Growth curve analysis
 performed using liquid culture absorbance at 526 nm
 readings are taken at regular intervals of time
 log phases of growth are estimated as the linear portion of the
log of absorbance versus time plot
 the specific growth rate gave a clear idea about the relative
toxicity of each of the amended metalloid
 lower specific growth rates suggest higher toxicity
•
•
•
•
Lag phase ( where the bacteria gets used to the new environment)
Log phase (growth phase of bacteria)
Stationary phase (no growth)
Death phase
www.bioc.rice.edu/.../NDL Bioreactor%20Page.htm
Growth Curve Results
0
0
1
2
3
4
5
6
7
8
LN of Optical Density
-0.5
-1
control
Selenite
Selenate
-1.5
Selenocyanate
Tellurite
-2
-2.5
-3
-3.5
Time (hrs)
Figure 1: Growth Curve for LHVE with 5 mM metalloid amendment.
0
0
1
2
3
4
5
6
7
8
LN of Optical Density
-0.5
-1
control
-1.5
Selenite
Selenate
-2
Selenocyanate
-2.5
Tellurite
-3
-3.5
Time (hrs)
Figure 2: Growth Curve for LHVE with 10 mM metalloid amendment.
Zone of Inhibition
 second method of estimating the relative toxicity
 it is the clear region around the paper disc saturated with metalloid
solution on the agar surface
 this is an indication of the absence, or the effective inhibition,
of microbial growth by the metalloid
 zone of inhibition of 52 mm was observed for tellurite amended plate
 tellurite was proved to be more highly toxic than all selenium anions
 these set of experiments further confirmed the growth curve results
Zone of Inhibition of LHVE
at 25 mM tellurite & 100 mM selenium anions
tellurite
control
selenite
selenate
selenocyanate
Headspace Analysis

part of the bioreduction process involves methylating Se

the headspace of the bacteria is sampled using solid-phase
microextraction fiber (SPME)
 fiber thickness is 75 µm (larger the surface area, the greater
the adsorption)

fiber exposure time is about 20-45 minutes.

splitless injection of sample in 2750C injector.

temperature Program: 300C for 2 minutes, ramped 150/min and
held at 2750C for 5 minutes.
What do you mean by headspace?
G = the gas phase (headspace)
The gas phase referred to as the
headspace and lies above the condensed
sample phase
S = the sample phase
The sample phase contains the
compound(s) of interest which are
volatile in nature that diffuse into the gas
phase until equilibrium is attained
Ref:duiblog.arizonaduicenter.com/tags/defense/
Solid Phase MicroExtraction
 rapid, simple, sensitive,
solvent-free extraction technique
 works on adsorption and
desorption principle
 concentrate the headspace
gases
Ref: www.chem.sc.edu/.../lab/images/RGFig1.JPG
DMeTS, 12.6
DMeDS, 8.78
MeSH, 2.55
Headspace Results
Chemiluminescence Intensit y
MeSH- methanethiol
DMeDS- dimethyl disulfide
DMeTS- dimethyl trisulfide
0
5
10
15
20
20
Time (min)
Figure 3: Chromatogram of LHVE control after 48 h.
DMeDSeS, 15.64
DMeDS, 8.78
DMeSeS, 10.09
DMeDSe, 11.29
DMeTS, 12.67
DMeSe, 5.58
MeSH, 2.63
MeSH- methanethiol, 2.63
DMeSe- dimethyl selenide, 5.58
Chemiluminescence Intensit y
DMeDS- dimethyl disufide, 8.78
e, 17.3417.34
DMeTSDMeTSe,
DMeSeDS, 13.68
DMeSeS- dimethyl selenenyl sulfide, 10.09
DMeDSe- dimethyl diselenide, 11.29
DMeTS- dimethyl trisulfide, 12.67
DMeSeDS- dimethyl selenenyl disulfide, 13.68
DMeDSeS- dimethyl diselenenyl disulfide, 15.64
DMeTSe- dimethyl triselenide, 17.34
0
5
10
15
20
Time (min)
Figure 4: Chromatogram of LHVE amended with 1.0 mM selenite, after 48 h.
DMeTS, 12.66
DMeDS, 8.76
MeSH, 2.60
Chemiluminescence Intensit y
MeSH- methanethiol, 2.60
DMeDS- dimethyl disufide, 8.76
DMeTS- dimethyl trisulfide, 12.66
0
5
10
15
20
Time (min)
Figure 5: Chromatogram of LHVE amended with 1 mM tellurite, after 48 h.
Table of Retention Times of Headspace compounds in GC-SCD
Compound
Formula
Boiling Point
(0C)
Retention Time (min)
Methanethiol
CH3SH
6
2.63
Dimethyl selenide
CH3SeCH3
58
5.58
Dimethyl disulfide
CH3SSCH3
110
8.78
Dimethyl selenenyl sulfide
CH3SeSCH3
131
10.09
Dimethyl diselenide
CH3SeSeCH3
153
11.29
Dimethyl trisulfide
CH3SSSCH3
170
12.67
Dimethyl selenenyl disulfide
CH3SeSSCH3
190
13.68
Dimethyl diselenenyl
sulfide
Dimethyl triselenide
CH3SeSeSCH3
217*
15.64
CH3SeSeSeCH3
236*
17.34
Abundance
GC-MS Results
Time (min)
Figure 6: Total ion chromatogram of an empty SPME fiber.
Abundance
1- Butanamine
Butamine
To be confirmed, may be from
From
SPME the
fiber SPME fiber
Time (min)
Figure 7: Total ion chromatogram of LHVE control after 72 h.
DMeSeS- dimethyl selenenyl sulfide, 6.3
Abundance
DMeDSe- dimethyl diselenide, 7.32
DMeSeDS- dimethyl selenenyl disulfide, 9.47
*DMeDSeS- dimethyl diselenenyl disulfide, 10.38
6
e S,
S
e
DM
.3
*DMeTSe- dimethyl triselenide, 11.17
DMeDSe, 7.32
DMeSeDS, 9.47
DMeSeDS, 9.41
* TWO NEW COMPOUNDS
*DMeDSeS, 10.38
*DMeTSe, 11.17
Time (min)
Figure 8: Total ion chromatogram of LHVE amended with selenite after 72 h.
93
m/z
Abundance
127
222
110
80
175
190 207
216
160
142
184
Fragment
80
Se
93
CH3-Se-
110
CH3-Se-CH3
127
CH3-Se-S-
142
CH3-Se-S-CH3
160
-Se-Se-
175
CH3-Se-Se-
190
CH3-Se-Se-CH3
207
CH3-Se-Se-S-
216
CH3-Se-Se-S-CH3
222
CH3-Se-Se-S-CH3
m/z
Figure 9: Mass spectrum of dimethyl diselenenyl sulfide at 10.38 min.
95
Abundance
m/z
175
160
80
270
190
Fragment
80
Se
95
CH3-Se-
160
-Se-Se-
175
CH3-Se-Se-
190
CH3-Se-Se-CH3
255
CH3-Se-Se-Se-
270
CH3-Se-Se-Se-CH3
255
m/z
Figure 14: Mass spectrum of dimethyl triselenide at 11.17 min.
Conclusions
 amendments had pronounced effect on the specific growth rate (SGR)
of LHVE
TeO32- > > SeO32- > SeO42- = SeCN-
 zone of inhibition experiments, further confirmed the SGR results
 headspace analysis showed a diverse production of organo-sulfur
and -selenium containing volatiles, but no organo-tellurium
 identification of two new compounds: DMDSeS, DMTSe
Acknowledgements
• Department of Chemistry, Sam Houston State University
• Ms. Rachelle Smith,
Analytical Laboratory Manager, TRIES Lab
• Funding from Robert A. Welch Foundation
• Rekha Raghavendra, for guiding in toxicity experiments
• Dr. Stacey Edmonson, UWGRE
Thank You…
Questions????